锌指核酶技术的原理及应用

锌指核酶(zinc finger nucleases,ZFNs)是将锌指蛋白的DNA识别域和非特异性核酸内切酶FokⅠ人工连接而构成的一种核酶.1对ZFNs能在DNA上产生双链断裂(double-strand breaks,DSB),诱导细胞发生同源重组(homology recombination,HR)或非同源末端连接(nonhomologous end joining,NHEJ).最近锌指核酶技术在基因功能的研究中受到重视.作者就ZFNs的作用原理、关键技术及其应用领域进行介绍.     

锌指核酶技术的原理及应用

Zinc finger nuclease (ZFN), which is a chimeric fusion structure between a Cys2-His2 zinc-finger protein (ZFP) and the cleavage domain of Fok Ⅰ endonuclease,can be used to introduce targeted double-stranded breaks (DSBs). ZFN-mediated cleavage leads to mutations when double-stranded breaks are repaired by homologous recombination (HR) or nonhomologous end joining (NHEJ). In recent years, ZFNs are widely used in the fields of genetic research. In this review, the methodology and technical advantages of ZFNs were briefly discussed.     

核酶技术简介

80年代初美国科罗拉多大学博尔分校的 Thomas Cech和耶鲁大学的 Sidney Alyt-man各自独立地发现具有生物催化功能的RNA,即核酶 ribozyme。核酶是一种能序列特异性地剪切 RNA的 RNA分子。很好设计的核酶能与被选择的 m RNA片段特异性地结合 ,这种结合能够通过多种机制特异地阻断 m RNA的表达。因此 ,它对于 RNA结构的研究以及异常基因表达所致疾病的治疗具有重要意义。近年来 ,核酶领域以令人惊讶的速度不断发展 ,大量文献得以积累 ,本文旨在对几年来的进展作一简要的回顾。1　核酶的结构目前研究得较多的有两种核酶 ,锤头核酶 (ham…     

脱氧核酶及其应用进展

脱氧核酶是利用体外分子进化技术合成的一种具有催化功能的单链DNA片段 ,具有高效的催化活性和结构识别能力。迄今为止 ,已发现大量具有催化功能的脱氧核酶。其中具有RNA切割活性的脱氧核酶 ,能催化RNA特定部位的切割反应 ,从mRNA水平对基因灭活 ,从而调控蛋白质的表达 ,可能成为治疗肿瘤、病毒感染性疾病以及其它相关疾病 ,基因功能研究 ,核酸突变分析等的新型工具。     

基因沉默技术在斑马鱼研究中的应用

模式生物斑马鱼主要应用于探索遗传学、发育生物学和分子生物学等研究领域的分子机制,常用的研究策略是DNA诱变和基于RNA的基因沉默.但化学诱变技术和传统反义核酸技术存在缺陷,目前在应用中已受到很大限制.反义morpholino技术具有稳定、方便的特点,广泛应用于斑马鱼基因功能的研究.新兴的锌指核酶基因敲除技术不依赖于胚胎干细胞使其前景更为广阔.     

脱氧核酶在肿瘤基因治疗中的应用

脱氧核酶是利用体外分子进化技术获得的一种具有酶活性的DNA分子。概述了脱氧核酶的结构特点、修饰和设计策略、催化特点、在基因治疗中的优势及作为一种新型的基因治疗工具在肿瘤治疗中的应用。     

发夹状核酶研究进展

发夹状核酶是一种具有催化功能的 RNA,能够特异识别并切割底物。它的切割效率较高 ,对金属离子和 p H值变动的依赖较小。在抗病毒和抗疾病相关基因表达的基因治疗领域有很高应用前景。本文对它的结构、反应动力学、催化机理和应用方面的研究进展作一综述     

核酶抑制乙型肝炎病毒的研究进展

核酶是一种有自动切割作用的RNA分子,不仅能够与双链DNA或者是RNA分子结合从而抑制他们的转录或翻译,而且由于它的自动切割作用能够在相应的位点切割核酸序列,具有酶的活性,因而其在HBV的基因治疗中的前景比较看好。本文针对乙型病毒性肝炎核酶的设计、构建、靶向性导入和稳定表达等问题进行了综述。     

脱氧核酶的研究进展

脱氧核酶是近年来利用体外分子进化技术合成的一种具有催化功能的单链 DNA片段 ,它能催化 RNA特定部位的切割反应 ,从 m RNA水平对基因灭活 ,从而调控蛋白的表达 ,可能成为对抗 RNA病毒感染、肿瘤等疾病的新型工具     

核酶在病毒性感染基因治疗中的应用

核酶与底物ＲＮＡ结合后行使其切割功能，可用于降特定的ｍＲＮＡ，控制多种病毒感染以及用于癌症的治疗。ＲＮＡ在病毒和其他疾病中具有重要的作用，针对ＨＩＶ基因组的基本结构，利用核酶特异性降解靶ＲＮＡ的特性，人们成功地设计对针对ＨＩＶ－１不同结构ＲＮＡ的核酶并成功地介导了核酶基因转入ＣＤ４＾＋细胞或细胞株的基因治疗。除ＡＩＤＳ外，核酶还成功地用于抗ＨＢＶ，ＨＰＶ等病毒性感染的研究中。     

锌指基因217与肿瘤

锌指基因217(zinc finger gene217,ZNF217)定位于染色体20q13.2,在各种肿瘤如乳腺癌、卵巢癌、胃癌中过度扩增,且与肿瘤的浸润性关系密切。ZNF217的过度扩增能减弱由端粒功能障碍和阿霉素引起的凋亡信号,引起Akt的磷酸化增加,促进肿瘤的发生和生长。抑制ZNF217能增加细胞对阿霉素的敏感性。ZNF217是一转录阻遏蛋白,能募集CtBP1/2并通过其锌指结构与启动子相结合,调节基因的转录。这些基因与细胞分化和增殖有关。目前由于缺乏明确的ZNF217靶向基因,ZNF217的功能尚不完全清楚。     

一个新的锌指蛋白基因ZNF333 cDNA的分离及表达谱分析

本研究通过生物信息学和分子生物学相结合的方法成功克隆了一个新的锌指蛋白基因ZNF333 cDNA。ZNF333基因包含14个外显子，编码665个氨基酸。蛋白质N端包含2个保守的KRAB－A盒，2个多态的KRAB－B盒，而C端包含10个重复的C2H2型锌指基序。ZNF333的表达在心脏组织中最高，在其他组织中也可检测到表达。同时还发现该基因存在选择性剪切形式。通过与人类基因组框架图的比较将ZNF333定位在19p13.1。     

设计特异性识别HBV核心启动子的锌指蛋白以抑制HepG2.2.15细胞中HBV的转录

目的:设计特异性结合乙肝病毒核心启动子(HBVCp)的锌指蛋白(ZFP),观察ZFP在体外对HBV转录的抑制情况。方法:利用Zinc finger tools设计特异性结合HBV Cp的ZFP。将ZFP核苷酸序列人工合成后克隆至pEGFP-N1和pcDNA3.1(+),构建真核表达质粒pEGFP-N1/ZFP-Flag和pcDNA3.1(+)/ZFP。通过倒置荧光显微镜、RT-PCR和Western blot鉴定pEGFP-N1/ZFP-Flag在COS-7中的表达情况;将pcDNA3.1(+)/ZFP转入HepG2.2.15细胞后24 h,采用ELISA和FQ-PCR检测上清液中HBeAg和HBV DNA含量,RT-PCR检测细胞内HBV mRNA水平。结果:ZFP在COS-7细胞中能正常表达。与空质粒组相比,ZFP组细胞培养上清液中HBV DNA拷贝量和HBeAg含量明显降低(P<0.05),细胞内HBV mRNA也显著减少。结论:人工设计的ZFP可以在真核细胞内正常表达,并能有效抑制HBV的转录。     

Different nuclear/cytoplasmic distributions of RET finger protein in different cell types

The RET finger protein (RFP), which belongs to the B box zinc finger protein family, has a tripartite motif consisting of a Ring finger, a B box finger and a coiled-coil domain. The RET finger protein becomes oncogenic when its tripartite motif is fused with the tyrosine kinase domain of the RET protein. This study examined the RFP expression in normal and tumor tissues by immunohistochemistry. RFP was detected in the nuclei of various cells, including peripheral and central neurones, hepatocytes, adrenal chromaffin cells and male germ cells. Among them, RFP was expressed at high levels in male germ cells such as primary spermatocytes and round spermatids, and formed a perinuclear cap structure in primary spermatocytes. On the other hand, high levels of cytoplasmic expression of RFP were observed in some plasma cells as well as solitary plasmacytoma and multiple myeloma. These results suggested that different nuclear/cytoplasmic distributions of RFP might play a role in the regulation of growth or differentiation of different cell types.     

ZEB基因在肿瘤发生发展中的作用

E盒结合锌指蛋白(zinc finger E-box binding homeobox,ZEB)是重要的细胞核转录因子之一,在肿瘤发生发展中发挥重要的作用。通过介导肿瘤细胞的黏附性、细胞极性、细胞凋亡,调控原癌基因和维持微环境等过程,促进肿瘤的发生及发展。在肿瘤治疗中可能成为一个新的靶向基因。     

Zinc finger protein x-linked (ZFX) contributes to patient prognosis,cell proliferation and apoptosis in human laryngeal squamous cell carcinoma

Zinc finger protein, X-linked (ZFX) gene locus on the human X chromosome is structurally similar to the zinc finger protein, Y-linked gene. Its role in human laryngeal squamous cell carcinoma (LSCC) is still not clearly defined. This study was focused on investigating the role of zinc-finger protein X-linked (ZFX) in human LSCC. Expression levels of ZFX were examined in LSCC tissues, corresponding adjacent non-tumoral tissues and vocal leukoplakia tissues by immunohistochemistry (IHC). The association with the expression level of ZFX and LSCC clincopathological parameters was analyzed. The prognostic value of ZFX expression was also analyzed. Lentivirus-mediated RNA interference was applied to silence ZFX expression and the effects of ZFX knockdown on the growth of human LSCC primary cells was investigated. Overexpression of ZFX was found in LSCC tissues. The expression of ZFX was associated with the clinical stage of LSCC. Patients with higher level of ZFX experienced a poorer prognosis compared to those with lower level of ZFX. Knockdown of ZFX inhibited cell proliferation, colony formation and migration of LSCC primary cells. Moreover, ZFX silencing induced cell apoptosis. These results provide the convincing evidence for the first time that ZFX plays an important role in LSCC development and could be a potential therapeutic target or prognostic predictor for LSCC.     

Zinc finger of the cerebellum 5 promotes colorectal cancer cell proliferation and cell cycle progression through enhanced CDK1/CDC25c signaling

IntroductionColorectal cancer (CRC), mostly caused by external or environmental factors, is the third most common and lethal cancer worldwide. Although a large number of investigations have been carried out to reveal the evolution of CRC, the underlying mechanisms of CRC remain unclear.Material and methodsExpression of zinc finger of the cerebellum 5 (ZIC5) in CRC tissues and cell models was measured by qRT-PCR and IHC. Cell transfection was carried out for ZIC5 overexpression or knockdown. The MTT assay was applied to examine the capacity of glioma cell proliferation. Wound healing assay and tumor invasion assay were used to test the capacity of glioma cell migration and invasion respectively. Cell cycle analysis and western blot were used to verify the apoptosis rates of CRC cells upon ZIC5 overexpression or downregulation. A further tumor Xenograft study was used to examine the effects of ZIC5 on tumor malignancy in vivo.ResultsCell models using HCT116 and SW620 cells were established to study the ZIC5 function upon ZIC5 overexpression of knockdown. Consistently, we discovered that ZIC5 also significantly increased in Chinese CRC patients. In addition, ZIC5 promoted CRC cell proliferation through increasing the proportion of cells maintained in the S phase. ZIC5 overexpression facilitated the capacity of CRC cell migration and invasion. Inhibition of ZIC5 mitigated such malignant effects.ConclusionsCollectively, investigations of the ZIC5 in CRC provided a new insight into CRC diagnosis, treatment, prognosis and next-step translational therapeutic developments from bench to clinic.